Many peptides and peptide hormones are involved in the body's regulation of a normal, healthy metabolic state. Central to many metabolic diseases and disorders is the regulation of insulin levels and blood glucose levels. A number of hormones that lower blood glucose levels are released from the gastrointestinal mucosa in response to the presence and adsorption of nutrients in the gut.
Insulin secretion is modulated in part by secretagogue hormones, termed incretins, which are produced by enteroendocrine cells. The incretin hormone, glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by intestinal cells that has been shown to produce an enhancing effect on insulin secretion. GLP-1 is processed from proglucagon in the gut and enhances nutrient-induced insulin release (Krcymann, et al. (1987) Lancet 2:1300-1303). GLP-1 and various truncated forms of GLP-1, such as GLP-1 (7-36) amide, and GLP-1 (7-37) acid, are known to stimulate insulin secretion (insulinotropic action) and cAMP formation (see, e.g., Mojsov (1992) Int. J. Pep. Pro. Res. 40:333-343, Gutniak et al. (1992) New Eng. J. of Med. 326:1316-1322, Nauck et al. (1993) Diabetologia 36:741-744; Nauck et al., (1993) J. Clin. Invest. 91:301-307, and Thorens et al. (1993) Diabetes 42:1219-1225). GLP-1 (7-37) acid is C-terminally truncated and amidated to form GLP-1 (7-36) NH2. The biological effects and metabolic turnover of the free acid GLP-1(7-37) OH, and the amide GLP-1(7-36) NH2, are indistinguishable.
GLP-1(7-36) amide exerts a pronounced antidiabetogenic effect in insulin-dependent diabetics by stimulating insulin sensitivity and by enhancing glucose-induced insulin release at physiological concentrations. When administered to non-insulin dependent diabetics, GLP-1(7-36) amide stimulates insulin release, lowers glucagon secretion, inhibits gastric emptying and enhances glucose utilization. The use of GLP-1 type molecules for prolonged therapy of diabetes has been complicated because the serum half-life of such peptides is quite short.
The plasma half-life of active GLP-1 is <5 minutes, and its metabolic clearance rate is about 12-13 minutes. The major protease involved in the metabolism of GLP-1 is dipeptidyl peptidase (DPP) IV (CD26) which cleaves the N-terminal His-Ala dipeptide, thus producing metabolites, GLP-1 (9-37) OH or GLP-1 (9-36) NH2, which are variously described as inactive, weak agonists or antagonists of GLP-1 receptor. The stimulation of GLP-1 receptor by GLP-1(7-37) OH or GLP-1(7-36)NH2 results in adenylate cyclase activation, cAMP synthesis, membrane depolarization, rise in intracellular calcium and increase in glucose-induced insulin secretion.
Other important effects of GLP-1 on glucose homeostasis are suppression of glucagon secretion and inhibition of gastric motility. GLP-1 inhibitory actions on pancreatic alpha cell secretion of glucagon leads to decreases in hepatic glucose production via reduction in gluconeogenesis and glycogenolysis. This antiglucagon effect of GLP-1 is preserved in diabetic patients. Central effects of GLP-1 include increases in satiety, coupled with decreased food intake.
Native human GIP (gastric inhibitory peptide or glucose-dependent insulinotropic peptide), related to GLP-1, is a single 42 amino acid peptide synthesized in and secreted by specialized enteroendocrine K-cells. These cells are found throughout the intestine, concentrated primarily in the duodenum and proximal jejunum. The main stimulant for GIP secretion is ingestion of carbohydrate and lipid-rich meals. Following meal ingestion, circulating plasma GIP levels increase 10- to 20-fold. The half-life of intact GIP is estimated to be approximately 7.3 minutes in healthy subjects and 5.2 minutes in diabetic subjects. In serum, GIP is degraded by DPP-IV. The resulting short biological half-life limits the therapeutic use of GIP.
GIP stimulates insulin secretion in the presence of elevated glucose concentrations. Thus, the effect of endogenously released GIP appears to be an important mechanism of postprandial insulin secretion and does not appear to play a role in the fasting state. GIP stimulates beta-cell proliferation and cell survival in INS-1 islet cell line studies. Further, unlike GLP-1, GIP appears to act by accelerating emptying of the stomach rather than by inhibiting gastrointestinal motility.
Another family of peptide hormones implicated in metabolic diseases and disorders is the amylin family of peptide hormones, including amylin, calcitonin, calcitonin gene related peptide, adrenomedullin, and intermedin (also referred to as AFP-6). See, for example, Wimalawansa (1997) Crit Rev Neurobiol. 11:167-239. Amylin regulates the rate of glucose appearance in circulation through a slowing of gastrointestinal motility and gastric emptying and through suppression of postprandial glucagon secretion and food intake. Amylin has been shown to reduce weight or weight gain. Amylin may also be used to treat pain, bone disorders, gastritis, to modulate lipids, in particular triglycerides, or to affect body composition such as the preferential loss of fat and sparing of lean tissue. Calcitonin (CT) was named for its secretion in response to induced hypercalcemia and its rapid hypocalcemic effect. CT has an effect on plasma calcium levels and inhibits osteoclast function and is widely used for the treatment of osteoporosis. Therapeutically, salmon CT (sCT) appears to increase bone density and decrease fracture rates with minimal adverse effects.
Calcitonin gene related peptide (CGRP) is a neuropeptide whose receptors are widely distributed in the body, including the nervous system and the cardiovascular system. This peptide seems to modulate sensory neurotransmission and is a potent endogenous vasodilatory peptide. Reported biological effects for CGRP include: modulation of substance P in inflammation, nicotinic receptor activity at the neuromuscular junction, stimulation of pancreatic enzyme secretion, a reduction of gastric acid secretion, peripheral vasodilation, cardiac acceleration, neuro-modulation, regulation of calcium metabolism, osteogenic stimulation, insulin secretion, an increase in body temperature, and a decrease in food intake. An important role of CGRP is to control blood flow to various organs by its potent vasodilatory actions. Prolonged infusion of CGRP into patients with congestive cardiac failure has shown a sustained beneficial effect on hemodynamic functions without adverse effects, suggesting a use in heart failure. Other indications of CGRP use include renal failure, acute and chronic coronary artery ischemia, treatment of cardiac arrhythmia, other peripheral vascular disease such as Raynaud's phenomenon, subarachnoid hemorrhage, hypertension, and pulmonary hypertension.
Adrenomedullin (ADM) has effects on the cardiovascular system, cellular growth, the central nervous system and the endocrine system, with a range of biological actions including vasodilation, cell growth, regulation of hormone secretion, and natriuresis. Hinson et al. (2000) Endocrine Reviews 21:138-167. ADM affects such endocrine organs as the pituitary, the adrenal gland, reproductive organs and the pancreas. The peptide appears to have a role in inhibiting ACTH release from the pituitary. In the adrenal gland, it appears to affect the secretory activity of the adrenal cortex and it increases adrenal blood flow, acting as a vasodilator in the adrenal vascular bed. ADM has been shown to be present throughout the female reproductive tract and plasma levels are elevated in normal pregnancy. In the pancreas, ADM most likely plays an inhibitory role since it attenuated and delayed insulin response to an oral glucose challenge, resulting in initial elevated glucose levels. ADM can also affect renal function. A bolus administered peripherally can significantly lower mean arterial pressure and raise renal blood flow, glomerular filtration rate and urine flow. In some cases, there is also an increase in Na+ excretion.
Studies have also demonstrated that ADM acts on fetal and adult rodent osteoblasts to increase cell growth comparable to those of known osteoblast growth factors such as transforming growth factor-β. In the lung, ADM not only causes pulmonary vasodilation, but also inhibits bronchoconstriction induced by histamine or acetylcholine. In healthy volunteers, i.v. infusion of ADM has been shown to reduce arterial pressure and to stimulate heart rate, cardiac output, plasma levels of cAMP, prolactin, norepinephrine and rennin. In these patients, there was little or no increase in urine volume or sodium excretion observed. In patients with heart failure or chronic renal failure, i.v. ADM had similar effects to those seen in normal subjects, and also induced diuresis and natriuresis, depending on the dose administered (Nicholls et al. (2001) Peptides 22:1745-1752). Experimental ADM treatment has also been shown to be beneficial in arterial and pulmonary hypertension, septic shock and ischemia/reperfusion injury (Beltowski (2004) Pol. J. Pharmacol. 56:5-27). Other indications for ADM treatment include: peripheral vascular disease, subarachnoid hemorrhage, hypertension, preeclamptic toxemia of pregnancy and preterm labor, and osteoporosis.
Expression of AFP-6 (i.e., intermedin) is primarily in the pituitary and gastrointestinal tract. In in vivo studies, AFP-6 administration led to blood pressure reduction in both normal and spontaneously hypertensive rats. In vivo administration in mice led to a suppression of gastric emptying and food intake (Roh et al. (2004)J. Biol. Chem. 279:7264-7274.)
Yet another peptide hormone family implicated in metabolic diseases and disorders is the leptin family. Leptin is an afferent signal in a negative feedback loop regulating food intake and body weight and the mature form of circulating leptin is a 146-amino acid protein.
Another peptide hormone implicated in metabolic diseases and disorders is cholecystokinin (CCK). Reported biological actions of CCK include stimulation of pancreatic secretion and pancreatic growth, stimulation of gallbladder contraction, delayed gastric emptying, inhibition of gastric acid secretion, stimulation of intestinal motility, stimulation of insulin secretion, and vasodilation. The actions of CCK also reportedly include effects on cardiovascular function, respiratory function, neurotoxicity and seizures, cancer cell proliferation, analgesia, sleep, sexual and reproductive behaviors, memory, anxiety and dopamine-mediated behaviors. It has also been reported that CCK in physiological plasma concentrations inhibits increases satiety and inhibits food intake in both lean and obese humans. See, for example, Lieverse et al. (1994) Ann. N.Y. Acad. Sci. 713:268-272, Crawley et al. (1994) Peptides 15:731-755, Walsh, “Gastrointestinal Hormones,” In Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Press, New York).
Another family of peptide hormones implicated in metabolic diseases and disorders is the pancreatic polypeptide family (PPF). The PPF includes pancreatic polypeptide (PP), Peptide YY (PYY), and Neuropeptide Y (NPY). These three related peptides have been reported to exert various biological effects. Effects of PP include inhibition of pancreatic secretion and relaxation of the gallbladder, and centrally administered PP produces modest increases in feeding that may be mediated by receptors localized to the hypothalamus and brainstem (reviewed in Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22 (1998)). Peripheral administration of PYY reportedly reduces gastric acid secretion, gastric motility, exocrine pancreatic secretion, gallbladder contraction, and intestinal motility (Yoshinaga et al. (1992) Am. J. Physiol. 263:G695-701, Guan et al. (1991) Endocrinology 128: 911-916, Pappas et al. (1986) Gastroenterology 91:1386-1389, Savage et al. (1987) Gut 28:166-170). The effects of central administration (injection in or around the hindbrain/brainstem) of PYY on gastric emptying, gastric motility and gastric acid secretion may differ from those effects observed after peripheral injection (Chen et al. (1995) Am. J. Physiol. 269: R787-792, Chen et al. (1986) Regul. Pept. 61:95-98 (1996) Yang et al. (1995) Am. J. Physiol. 268: G943-948, Chen et al. (1997) Neurogastroenterol. Motil. 9:109-116). PYY has been shown to stimulate food and water intake after central administration (Morley et al. (1985) Brain Res. 341:200-203, Corp et al. (1990) Am. J. Physiol. 259: R317-323).
Metabolic diseases and disorders take on many forms, including obesity, diabetes, dyslipidemia, and insulin resistance. Obesity and its associated disorders are common and very serious public health problems in the United States and throughout the world. Upper body obesity is the strongest risk factor known for type 2 diabetes mellitus and is a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovarian syndrome, cancers of the breast, prostate, and colon, and increased incidence of complications of general anesthesia (see, e.g., Kopelman (2000) Nature 404:635-643).
Obesity reduces life-span and carries a serious risk of the co-morbidities listed above, as well disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholelithiasis, orthopedic injury, and thromboembolic disease (Rissanen et al. (1990) Br. Med. J. 301:835-837). Obesity is also a risk factor for the group of conditions called insulin resistance syndrome, or “Syndrome X” and metabolic syndrome. The worldwide medical cost of obesity and associated disorders is enormous. The pathogenesis of obesity appears to be multifactorial but the basic problem is that in obese subjects nutrient availability and energy expenditure do not come into balance until there is excess adipose tissue. Obesity is currently a poorly treatable, chronic, essentially intractable metabolic disorder. A therapeutic drug useful in weight reduction of obese persons could have a profound beneficial effect on their health.
Diabetes is a disorder of carbohydrate metabolism characterized by hyperglycemia and glucosuria resulting from insufficient production or utilization of insulin. Diabetes severely affects the quality of life of large parts of the populations in developed countries. Insufficient production of insulin is characterized as type 1 diabetes and insufficient utilization of insulin is type 2 diabetes. However, it is now widely recognized that there are many distinct diabetes related diseases which have their onset long before patients are diagnosed as having overt diabetes. Also, the effects from the suboptimal control of glucose metabolism in diabetes give rise to a wide spectrum of related lipid and cardiovascular disorders.
Dyslipidemia, or abnormal levels of lipoproteins in blood plasma, is a frequent occurrence among diabetics. Dyslipidemia is typically characterized by elevated plasma triglycerides, low HDL (high density lipoprotein) cholesterol, normal to elevated levels of LDL (low density lipoprotein) cholesterol and increased levels of small dense, LDL particles in the blood. Dyslipidemia is one of the main contributors to the increased incidence of coronary events and deaths among diabetic subjects. Epidemiological studies have confirmed this by showing a several-fold increase in coronary deaths among diabetic subjects when compared with non-diabetic subjects. Several lipoprotein abnormalities have been described among diabetic subjects.
Insulin resistance is the diminished ability of insulin to exert its biologically action across a broad range of concentrations. In insulin resistance, the body secretes abnormally high amounts of insulin to compensate for this defect and a state of impaired glucose tolerance develops. Failing to compensate for the defective insulin action, the plasma glucose concentration inevitably rises, resulting in the clinical state of diabetes. It is being recognized that insulin resistance and relative hyperinsulinemia have a contributory role in obesity, hypertension, atherosclerosis and type 2 diabetes. The association of insulin resistance with obesity, hypertension and angina has been described as a syndrome, Syndrome X, having insulin resistance as the common pathogenic link.
Hypertension, or high blood pressure, may or may not be associated with a metabolic disease or disorder in a patient. Hypertension is the most common disease affecting the heart and blood vessels and statistics indicate that it occurs in more than 50 million Americans. The prevalence of hypertension increases with age. Hypertension is of considerable concern because of the harm it can do to the heart, brain and kidneys if it remains uncontrolled.
There remains a need for effective methods of producing bioactive peptides in forms that are effective for the treatment for diseases, conditions, and disorders, including metabolic and cardiovascular diseases and related disorders.
All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.